This invention relates to direct, specific binding assays for free ligands in test samples where the ligand is also found sequestered to endogenous receptors. This invention particularly relates to immunoassays for free thyroxine in test samples containing thyroxine binding proteins such as TBG and albumin.
Free ligands are defined for the purposes here as substances, usually of low molecular weight but not necessarily, which are present unbound in a test sample which contains an endogenous receptor for the ligand. The biological role for such receptors has not been definitively established; it is likely that they serve as carriers for the ligand, thereby creating a reservoir of the ligand which can dissociate by mass action as the free ligand is consumed in biological processes. Typically all but a small percentage of the ligand is present bound to its receptors.
Such receptors are typically proteins. The receptors may be specific, as in the case of autoantibodies, transcortin and TBG, or relatively nonspecific, for example albumin. The term endogenous does not mean that the receptor normally occurs in patient sample or is even of biological origin. It means only that the test sample as it is obtained from the patient is expected or potentially may contain a receptor which is binding some proportion of the ligand of interest.
Astute clinicians are turning increasingly to free ligand assay in an effort to more accurately assess the status of their patients. This effort has been facilitated by the appearance in the marketplace of so-called direct immunoassays for free ligands, more specifically free thyroxine immunoassays. Direct ligand assays are characterized by the measurement of the free ligand itself, rather than by other determinations which are then correlated to the free ligand concentration by calculations such as in the free thyroxine index.
Two techniques now exist for direct free ligand assay aside from equilibrium dialysis, a technique not particularly well suited to speedy and routine clinical laboratory use. In both techniques the free ligand is bound to a receptor included in the test kit and the amount of bound ligand is then determined. The methods differ in the manner in which the endogenous receptor present in the test sample is prevented from interfering in the assay. In the method of British Patent Application 2,030,290 the endogenous receptor is excluded by physically separating the test sample residue from the test receptor-bound ligand after adsorption of the free ligand from the test sample, i.e., by incubating test sample with insolubilized antibody for ligand, decanting the test sample residue, washing, adding tracer-labelled ligand and measuring the amount of tracer taken up by the antibody (which in turn is inversely proportional to the amount of ligand which bound to the test receptor). This method is commercially advantageous because it employs simple, pre-existing reagents. Radiolabelled thyroxine, which has been used for some time in total thyroxine tests, is satisfactory for use in the above-described direct free thyroxine assay as well. However, each ligand assay requires an analogue of that ligand and no labelled immuno reagent common to all ligand assays could be employed.
The direct free ligand assay described in European Patent Application 0 026 103 uses another system for neutralizing the potential interference by endogenous receptors. Rather than excluding the endogenous receptors from interaction with labelled ligand analogue by washing the receptors out of the system, the ligand analogue is chemically excluded from binding to the endogenous receptor. This is accomplished by using as ligand analogue a derivative of the ligand which binds insubstantially to endogenous receptor but which does bind comparatively well to test receptor. This derivative will for convenience be termed a differential binding ligand analogue hereinafter. This assay for free ligand has the advantage that it does not require an intermediate washing step. It does, however, require the special synthesis of a carefully designed tracer for each and every different ligand to be assayed.
Since it is believed that in the future a variety of ligands will be assayed by such direct methods, it would be commercially desirable to use common reagents among the assays if possible, the advantages including ease of manufacture, stability and improved assay performance.
Accordingly, it is an object of this invention to simplify reagent preparation in direct assays for free ligands.
The present invention provides a method for determining the amount of free ligand in a test sample where the ligand is also present bound to one or more endogenous receptors, comprising combining the test sample, ligand receptor and unlabelled differential binding ligand analogue, incubating to permit the free ligand and unlabelled, differential binding ligand analogue to compete for the ligand receptor separating the ligand analogue, determining the amount of ligand receptor bound to the ligand or to the ligand analogue, and correlating the amount of bound ligand receptor to the amount of free ligand present in the test sample.
The invention also resides in a composition for use in the assay of test samples containing free ligand and endogenous receptors for such ligand, comprising the ligand covalently conjugated to a substantially water insoluble substrate or to a water soluble substance physically adsorbed onto a substantially water insoluble substrate, said ligand being conjugated so as to substantially exclude the binding to such insoluble ligand of receptors for such ligand present in said test samples.
The invention, therefore, dispenses with the labelled, differential binding ligand analogue called for in the above-mentioned European Patent Application. Instead, the test receptor is directly or indirectly labelled. As will be explained below, it is now possible to contemplate a multipurpose or universal tracer, i.e. one which has common utility in all free ligand assays.
The assay method of this invention employs two principal reagents: First, the differential binding ligand analogue which is insolubilized or can be made so as part of the analytical procedure and, second, a labelled ligand antibody or an unlabelled ligand antibody conjugated to a labelled anti-(ligand antibody).
The differential binding ligand analogue comprises a residue of the ligand capable of binding anti-ligand antibody or other anti-ligand test receptor to the relative exclusion of endogenous receptor, which is covalently linked to a substantially water insoluble substance or, more conveniently, covalently linked to a substantially water soluble component which is (a) physically adsorbed onto an insoluble substrate or (b) further reacted to form a substantially water insoluble material.
The assay is conducted by combining the test sample, ligand antibody and unlabelled, differential binding ligand analogue, incubating to permit the free ligand and unlabelled, differential binding ligand analogue to compete for the ligand antibody, determining the amount of ligand antibody bound to the ligand or to the ligand analogue, and correlating the amount of bound ligand antibody to the amount of free ligand present in the test sample.